This invention relates to the discovery and uses of a class of short DNA aptamers that selectively recognize CD200R1, a protein expressed on the surface of myeloid and lymphoid cells that delivers immune inhibitory signals to modulate inflammation when engaged with its ligand, CD200.
CD200R1
CD200R1 is a type I glycoprotein expressed on cells of myeloid and lymphoid lineage. It delivers immune inhibitory signals upon ligation to the widely distributed cell surface glycoprotein CD200. Structurally, CD200R1 contains two Ig-like domains, a transmembrane region, and a cytoplasmic tail containing a NXPY motif which is phosphorylated upon CD200 ligation inducing recruitment of the adaptor protein Dok2 and subsequent signal transduction.
The physiological importance of CD200:CD200R1 inhibitory signalling has been established in a number of diseases including arthritis, transplantation, and a number of central nervous system autoimmune diseases such as Parkinson's disease (PD) and multiple sclerosis (MS). For instance, a recombinant CD200.Fc fusion protein has been shown to behave as a potent in-vivo immunosuppressant, prolonging allo- and xenograph survival as well as suppressing collagen-induced arthritis in mice. Also, the inhibition of CD200:CD200R1 signalling on microglial cells using a blocking antibody to CD200R1 exacerbated neurodegeneration and disease state in a murine model of experimental autoimmune encephalomyelitis (EAE). These findings were further supported in a separate EAE study where treatment with CD200.Fc suppressed microglial accumulation, and decreased the production of pro-inflammatory cytokines IL-6, TNF-α, and nitric oxide by myeloid cells in the spleen and central nervous system.
CD200R1 signalling has also been implicated in tissue specific autoimmunity, as both systemic and local treatment with an anti-CD200R1 agonistic antibody suppressed experimental autoimmune uveitis (EAU), a model of CD4+ T-cell organ-specific autoimmunity of the eye.
Thus the development of safe and effective immunomodulatory agents which stimulate CD200R1 signalling are of clinical interest. Despite advances in antibody and protein engineering, the major drawbacks of protein-based CD200R1 stimulators are their immunogenicity arising from their chronic use and their production costs resulting in expensive therapies for patients.
It would be useful to have a non-protein composition that binds to CD200R1 with high specificity and/or affinity without immune responses. Such a composition may act as a stimulator of immune inhibitory signalling by selectively binding, and hindering the function of, or inactivating, the CD200R1, and thus be useful as a treatment for immune related disease such as arthritis, asthma, allergy, infection, as a course of treatment during or after transplantation, or for treatment of autoimmune disorders such as systemic lupus erythematosus, Parkinson's Disease, or multiple sclerosis. Such a composition may also be useful conjugated or otherwise associated with a cytotoxic agent for specifically targeting such an agent to a CD200R1 expressing or overexpressing cell.
Aptamers
Aptamers are short, single-stranded nucleic acid oligomers (ssDNA or RNA) which adopt a specific tertiary structure allowing them to bind to molecular targets with high specificity and affinities comparable to that of monoclonal antibodies, through interactions other than classic Watson-Crick base pairing. In some cases, aptamers will display functional properties beyond just binding to their target. For instance, an aptamer to the inflammation factor human neutrophil elastase (hNE) was shown to significantly reduce lung inflammation in rats and displayed greater specificity for their target than an antielastase IgG control. Examples of other aptamers exhibiting functional attributes include a DNA aptamer to anti-HIV reverse transcriptase and RNA aptamers to the basic fibroblast growth factor and vascular endothelial growth factor. Finally, a single-stranded DNA aptamer selected to bind to thrombin has been shown to inhibit thrombin-catalyzed fibrin-clot formation in vitro using either purified fibrinogen or human plasma. Thus, aptamers can be derived to either block protein-protein interactions or act as agonists to cell surface receptors.
Aptamers have been generated for over 100 proteins including growth factors, transcription factors, enzymes, immunoglobulins, and receptors. A typical aptamer is 10-15 kDa in size (30-45 nucleotides), binds its target with sub-nanomolar affinity, and discriminates against closely related targets. They have several advantages over antibodies. As a class, they have demonstrated therapeutically acceptable toxicity, and a lack of immunogenicity. Aptamers can typically be administered by subcutaneous injection due to their low solubility as compared to antibodies. Aptamers are chemically robust, and can be readily manufactured since they can be chemically synthesized.
In contrast to antibodies and other protein-based agents, aptamers have a number of advantages including a long shelf life, low immunogenicity, and chemical synthesis. However, aptamers as therapeutic entities do display poor pharmacokinetic profiles as unprotected RNA or DNA aptamers are rapidly removed from circulation due to renal filtration and nuclease degradation. However, improved pharmacokinetic properties have been observed upon site-specific conjugation of polyethylene glycol (PEG) polymers to aptamer termini as well as the incorporation of nuclease resistant 2′-F or 2′-Me nucleotides in the case of RNA aptamers. Functional aptamers which target co-stimulatory or co-inhibitory receptors represent a new class of targeted immunotherapeutic agents with unique and advantageous properties. Thus far, aptamers with either agonistic or antagonistic function have been developed to a number of immune co-receptors including CTLA-4, 4-1BB, OX-40, IL-6R, IL-10R, and CD28. However, only a few of them have been validated for activity in-vivo.
Membrane impermeant aptamers have the potential to be used as antagonists themselves, or to serve as intracellular delivery agents specific to an internalized surface marker on a cancer cell, for example. Therapeutic cargos such as siRNAs, antisense oligonucleotides, ribozymes as well as low MW drugs, can be directly coupled to aptamers or packaged into particles modified with aptamers. Aptamer-containing conjugates can be constructed by chemically coupling a drug, such as a chemotherapeutic drug, to the aptamer via a linker or by intercalating the drug into the aptamer folded structure creating a physical complex. The cargo is then imported into a target cell due to the aptamer specificity while reducing toxicity towards other cells. Cargos can be conjugated to aptamers during solid-phase synthesis or post-synthesis by incorporating an amino or thiol group at one end of the oligonucleotide during its assembly. A therapeutic protein can also be coupled to the aptamer, to reach an intracellular substrate target. Aptamers can also be conjugated to radionuclides or metal chelators to image or kill cells targeted by the aptamer. Recently, aptamers have been conjugated to nanostructures, representing a promising class of new agents for targeted imaging and therapy. Thus, cargos can also be encapsulated into such nanopaticles decorated on their surface with aptamers. The targeted structures include nanorods, quantum dots as well as soft and hard nanoparticles.
An aptamer that binds with high specificity and/or affinity to CD200R1 would be desirable for its potential as a simple (compared, for example, to an antibody), synthetic, potentially non-immunogenic stimulator of immune inhibitory signalling. Such a compound would be useful for a variety of research, diagnostic, and therapeutic uses, for example, for imaging, diagnosis, or for the treatment of immune related disease such as arthritis, allergy, asthma, infection, as a course of treatment during or after transplantation, or for treatment of autoimmune disorders such as systemic lupus erythematosus, Parkinson's Disease. or multiple sclerosis. Such a compound could also be useful conjugated or otherwise associated with a cytotoxic agent for specifically targeting such an agent to a CD200R1-expressing or overexpressing cell.
Asthma
Asthma is a common long-term inflammatory disease of the airways of the lungs, affecting hundreds of millions of individuals, and causing hundreds of thousands of deaths each year. Symptoms of asthma include episodes of coughing, shortness of breath, wheezing, coughing, and difficulty breathing. Currently, the most common treatment for asthma is inhaled or intranasally administered corticosteroids. They may be combined with long-acting beta agonists, or antileukotriene agents. Effectiveness of these treatments is highly variable. The cause of asthma is also highly variable, and may include both environmental factors and genetic factors. Many of the genes associated or implicated with asthma are related to the immune system, or modulating inflammation.